Blood-brain barrier permeable peptide compositions

ABSTRACT

Blood-brain barrier permeable peptide compositions that contain variable antigen binding domains from camelid and/or shark heavy-chain only single-domain antibodies are described. The variable antigen binding domains of the peptide compositions bind to therapeutic and diagnostic biomarkers in the central nervous system, such as the amyloid-beta peptide biomarker for Alzheimer&#39;s disease. The peptide compositions contain constant domains from human IgG, camelid IgG, and/or shark IgNAR. The peptide compositions include heavy-chain only single-domain antibodies and compositions with one or more variable antigen binding domain bound to one or more constant domains.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.13/736,852, filed Jan. 8, 2013, and claims the benefit under 35 U.S.C. §119(e) of the U.S. Provisional Application 61/631,731, filed Jan. 9,2012, the contents of each of which is hereby incorporated by referencein its entirety into the present disclosure.

FIELD OF THE INVENTION

The invention relates to the discovery of blood-brain barrier (BBB)permeable peptide compositions and antibody-mimics derived from camelidand shark heavy-chain only antibodies, their analogs, and uses thereof.

BACKGROUND OF THE INVENTION A. The Blood-Brain Barrier

The blood-brain barrier (BBB) has been an impediment to successful drugdelivery to the central nervous system (CNS). As a consequence, mostdiseases of the brain cannot be diagnosed and treated. Typically, onlysmall-molecule drugs cross the BBB. That is why, for too long, theprocess of drug discovery has been centered on designing, developing andscreening small molecules with activity at a particular site or receptorin the brain. However, small-molecule drugs for CNS targets havelimitations, which include: i) non-specific targeting; ii) non-specificorgan distribution; iii) low therapeutic indices; iv) development ofdrug resistance shortly after initial treatment; v) only a smallpercentage of small-molecule drugs cross the BBB and vi) only 1% of thetotal number of drugs were active in the CNS [Pardridge W M, NeuroRX, 2(1), 3 (2006)]. In addition, only a few diseases of the brain, such asdepression, chronic pain, and epilepsy, respond to this category ofsmall molecules. Most serious diseases of the brain such as Alzheimer'sdisease (AD); Parkinson's disease (PD); brain cancer; stroke; brain andspinal cord injury; HIV infection of the brain; Huntington disease;multiple sclerosis (MS); and many childhood inborn genetic errorsaffecting the brain do not respond to small molecule drugs, irrespectiveof the lipid solubility of the drug. A handful of FDA approved smallmolecule drugs, e.g. Aricept (AD), Cognex (AD), Exelon (AD), Razadyne(AD), and Levodopa (PD), for neurodegenerative diseases that slow downthe disease symptoms in some patients stop working after a period oftime, leaving the patient to helplessly succumb to his/her disease.

Development of large molecule drugs is generally discouraged because oftheir typically poor BBB permeability. Many potential large moleculemodern drugs, such as, engineered proteins (e.g.: nerve growth factors),antibodies, genes, vectors, micro-RNA, siRNA, oligonucleotides andribozymes, which are otherwise effective in ex-vivo studies, have notbeen developed for clinical use due to a failure to deliver them insufficient quantity into the CNS. Although Alzheimer's disease (AD) hasbeen known for more than a century and despite enormous research effortsboth by private sectors and government institutes, there are nodiagnostics or curative treatments for diseases of the CNS. More than 55million people (and 6.5 million Americans in the US) are afflictedworldwide by neurodegenerative diseases (Alzheimer's disease andParkinson's disease are the most common forms of degenerative dementia).These troubling statistics demonstrate an unmet need of developingtechnologies to solve the issues of diagnosing and treatingneurodegenerative and tumor diseases in the CNS.

The BBB is formed by tight junctions between the cerebral endothelialcells, which are produced by the interaction of several transmembraneproteins that project into and seal the paracellular pathways (FIG. 1).The interaction of these junctional proteins, particularly, occludin andclaudin, is complex and effectively blocks an aqueous route of freediffusion for polar solutes from blood along these potentialparacellular pathways and thus denies these solutes free access tocerebrospinal fluid. Major scientific efforts over the years have led tothe development of the following methods to cross the BBB: (i) The useof liposomes or other charged lipid formulations, which have limitedcomplex stability in serum and high toxicity over time (Whittlessey K Jet al., Biomaterials, 27, 2477 (2006)); (ii) Electroporation-basedtechniques which are only effective when performed during a specificwindow of development in healthy cells, with eventual loss of expressionor bioactivity (Gartner et al., Methods Enzymology 406, 374 (2006)), and(iii) Viral-based vectors and fusions which have shown only limitedefficacy in humans and animals while raising a number of safetyconcerns, and typically requiring invasive procedures such as directinjection into the brain to achieve targeted delivery (Luo D, NatBiotechol, 18 (8), 893 (2000)). Thus, there is an unmet need to developnovel technologies to breach the BBB.

B. Strategies for Drug Delivery Across the Blood-Brain Barrier

Invasive strategies such as intra-cerebroventricular infusion,convection-enhanced delivery, and intra-cerebral Injection are coveredin the following references: Pardridge W M, Pharma Res., 24, 1733(2007); Pardridge W M, Neuro RX, 2, 3 (2005); Vandergrift W A, et al.,Neurosurg Focus, 20, E10 (2006); Funk L K, et al., Cancer Res., 58, 672(1998); Marks W J, et al., Lancet Neurol, 7, 400 (2008); and Herzog C D,et al., Mov. Disord, 22, 1124 (2007).

Disruption of the BBB using bradykynin analogues, ultrasound, andosmotic pressure are covered in the following references: Borlogan C V,et al., Brain Research Bulletin, 60, 2970306 (2003); Hynynen K, et al.,J. Neurosurg., 105, 445 (2006); and Fortin D, et al., Cancer, 109, 751(2007).

Physiological approaches involving transporter-mediated delivery,receptor-mediated transcytosis, adsorptive-mediated transcytosis arecovered in the following references: Allen D D, et al., J. Pharmacol ExpTher, 304, 1268 (2003); Coloma M J, et al., Pharm Res, 17, 266 (2000);Jones A R, et al., Pharma Res, 24, 1759 (2007); Boada R J, et al.,Biotech Bioeng, 100, 387 (2007); Pardridge W M, Pharma Res, 3, 90(2003); Zhang Y, et al., J. Pharmaco Exp Therap, 313, 1075 (2005); andZhang Y, et al, Brain Res, 1111, 227 (2006).

Pharmacological approaches involving chemical modification of drugs tolipophilic molecules or encapsulation into liposomes are covered by thefollowing references: Bradley M O, Webb N L, et al., Clin. Cancer Res.,7, 3229 (2001); Lipinski C A, Lombardo F, et al., Adv. Drug Deliv Rev,46, 3 (2001); Huwyler J, et al., J Pharmacol Exp Ther, 282, 1541 (1997);Madrid Y, et al., Adv Pharmacol, 22, 299 (1991); Huwyler J, Wu D, etal., Proc. Natl. Acad. Sci. USA, 93, 14164 (1996); Swada G A, et al., J.Pharmacol Exp Ther, 288, 1327 (1999); and Shashoua V E, et al., LifeSci., 58, 1347 (1996).

Resistance to opsonization and nanoparticles based drug delivery acrossthe BBB, whereby the drug is passively adsorbed on to the particles, iscovered by following references: Greiling W, Ehrlich P, Verlag E,Dusseldorf, Germany, p. 48, 1954; Couvreur P, Kante B, et al., J. PharmPharmacol, 31, 331 (1979); Douglas S J, et al., J Colloid. InterfaceSci, 101, 149 (1984); Douglas S J, et al., J. Colloid Interface Sci.,103, 154 (1985); Khanna S C, Speiser P, J. Pharm. Sci, 58, 1114 (1969);Khanna S C, et al., J. Pharm. Sci, 59, 614 (1970); Sugibayashi K, etal., J Pharm. Dyn, 2, 350 (1979b); Brasseur F, Couvreur P, et al.,Actinomycin D absorbed on polymethylcyanoacrylate: increased efficiencyagainst an experimental tumor, Eur. J. Cancer, 16, 1441 (1980); Widder KJ, et al., Eur. J Cancer, 19, 141 (1983); Couvreur P, et al., Toxicityof polyalkylcyanoacrylate nanoparticles II. Doxorubicin-loadednanoparticles, J. Pharma Sci, 71, 790 (1982); Couvreur P, et al.,Biodegradable polymeric nanoparticles as drug carrier for antitumoragents, Polymeric Nanoparticles and Microspheres, CRC Press, Boca Raton,pp. 27-93 (1986); Grislain L, Couvreur P, et al., Pharmacokinetics anddistribution of a biodegradable drug-carrier, Int. J. Pharm., 15, 335(1983); Mukherjee P, et al., Potential therapeutic applications of goldnanoparticles in BCLL, J. Nanobiotechnology, 5, 4 (2007); Maeda H andMatsumura Y, Tumoritropic and lymphotropic principles of macromoleculardrugs, Crit. Rev. Ther Drug Carrier Syst., 6, 193 (1989); Kattan, J etal., Phase I clinical trial and pharmacokinetic evaluation ofdoxorubicin carried by polyisohexylcyanoacrylate nanoparticles, Invest.New Drugs, 10, 191 (1992); Kreuter J, Naoparticles—A historicalperspective, Int. J. Pharm., 331, 1 (2007); Alyautdin R, et al.,Analgesic activity of the hexapeptide dalargin adsorbed on the surfaceof polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles. Eur.J. Pharm. Biopharm., 41, 44 (1995); Kreuter J, Alyautdin R, et al.,Passage of peptides through the blood-brain barrier with colloidalpolymer particles (nanoparticles), Brain Res., 674, 171 (1995);Alyautdin R N et al., Delivery of loperamide across the blood-brainbarrier with polysorbate 80-coated polybutylcyanoacrylate nanoparticles,Pharm. Res., 14, 325 (1997); Schroeder U, et al., Body distribution of³H-labeled dalargin bound to polybutylcyanoacrylate, Life Sci., 66, 495(2000); Alyautdin R N et al., Significant entry of tubocurarine into thebrain of rats by absorption to polysorbate 80-coatedpolybutyl-cyanoacrylate nanoparticles: an in situ brain perfusion study,J. Microencapsul., 15, 67 (1998); Gulyaev A E, Gelperina S E, et al.,Significant transport of doxorubicin into the brain with polysorbate80-coated nanoparticles, Pharm. Res., 16, 1564 (1999); Steiniger S C J,Kreuter J, et al., Chemotherapy of glioblastoma in rats usingdoxorubicin-loaded nanoparticles, Int. J Cancer, 109, 759 (2004);Hekmatara T, et al., Efficient systemic therapy of rat glioblastoma bynanoparticle-bound doxorubicin is due to antiangiogenic effects, Clin.Neuropath., 28, 153 (2009); Gelperina S E, et al., Toxicological studiesof doxorubicin bound to polysorbate 80-coated polybutylcyanoacrylatenanoparticles in healthy rats and rats with intracranial glioblastoma,Toxicol. Lett., 126, 131 (2002); Couvreur P, et al., J. Pharm. Sci, 71,790 (1982); Kreuter J, et al., Apolipoprotein-mediated transport ofnanoparticles-bound drugs across the blood-brain barrier, J. DrugTargeting, 10, 317 (2002); Davis S S, Biomedical appplications ofnanotechnology-implications for drug targeting and gene therapy,Tibtech, 15, 217 (1997); Moghimi S M, Szebeni J, Stealth liposome andlong circulating nanoparticles: Critical issues in pharmacokinetics,opsonization and protein-binding properties, Progress in Lipid Research,42, 463 (2003); Arvizo R R, et al., Modulating pharmacokinetics, tumoruptake and biodistribution by engineered nanoparticles, PLos One, 6,e24374 (2011); Kurakhmaeva K B, et al., Brain targeting of nerve growthfactor using polybutylcyanoacrylate nanoparticles, J Drug Targeting, 17,564 (2009); and Reukov V, et al., Proteins conjugated topolybutylcyanoacrylate nanoparticles as potential neuroprotectiveagents, Biotechnology and Bioengineering, 108, 243 (2010).

Shortcomings of Nanoparticles.

Although nanoparticles have made significant contributions to the fieldof medical sciences, most of the published studies have been conductedwith drugs non-covalently coated to nanoparticles, thereby perhaps notrealizing the full potential of nanomedicine.

C. Single-Domain Antibodies

In 1983 it was reported that the sera of camelid contained two differentkinds of immunoglobulin: conventional heterodimeric IgGs composed ofheavy and light chains, and unconventional IgGs without the light chains[Grover Y P, et al., Indian Journal of Biochemistry and Biophysics, 20,238 (1983)]. Grover et al. demonstrated the presence of three bandswhich were designated as IgM, IgA, and a broad heterogeneous bandcontaining a mixture of IgG complexes. One can speculate that the broadband these authors observed was due to the presence of mixture of normalIgG with a molecular weight (MW) of ˜160 KDa and heavy-chain IgG,without the light chain, with a MW of ˜80 KDa. However, since theseauthors did not use a proper sizing marker, the broad IgGs band couldnot be satisfactorily characterized.

Ungar-Waron et al. disclosed a SDS-PAGE analysis of camelid IgGs mixturetreated with and without 2-mercaptoethanol (2ME) [Israel J. Vet.Medicine, 43 (3), 198 (1987)]. In the absence of 2-ME, IgG-complex,obtained from camelid serum, dissociated into two components withapproximate molecular weight (MW) of 160 KDa (Conventional IgG) and ˜100KDa (New IgG) on SDS-PAGE. However, in the presence of 2-ME, three bandsof MW 55 KDa (gamma-like heavy-chain), 22 KDa (Light chain) and anadditional protein band of 42 KDa (now known as heavy-chain only camelidantibody band without the light chains) were seen.

Subsequently, Azwai et al. from University of Liverpool, UK,independently confirmed the presence of an additional IgG band incamelid serums with a molecular weight of 42 KDa by SDS-PAGEelectrophoresis under reducing conditions [Azwai, S. M., et al., J.Comp. Path., 109, 187 (1993)].

Hamers-Casterman et al. also reported similar findings, confirmingindependently the presence of 42 KDa IgG subclass in the sera ofcamelids upon SDS-PAGE analysis under reducing conditions[Hamers-Casterman et al., Nature, 363, 446 (1993) and U.S. Pat. No.6,005,079].

Thus, two types of antibodies exist in camels, dromedaries, and llamas:one a conventional hetero-tetramer having two heavy and two light chains(MW˜160 KDa), and the other consisting of only two heavy chains, devoidof light chains (MW˜80 to 90 KDa).

In addition to camelid antibodies having only two heavy chains anddevoid of light chains, a distinctly unconventional antibody isotype wasidentified in the serum of nurse sharks (Ginglymostoma cirratum) andwobbegong sharks (Orectolobus maculatus). The antibody was called the Ignew antigen receptors (IgNARs). They are disulfide-bonded homodimersconsisting of five constant domains (CNAR) and one variable domain(VNAR). There is no light chain, and the individual variable domains areindependent in solution and do not appear to associate across ahydrophobic interface [Greenberg A S, Avila D, Hughes M, Hughes A,McKinney E, Flajnik M F, Nature 374, 168 (1995); Nuttall S D, Krishnan UV, Hattarki M, De Gori R, Irving R A, Hudson P J, Mol. Immunol., 38, 313(2001), Comp. Biochem. Physiol. B., 15, 225 (1973)]. There are threedifferent types of IgNARs characterized by their time of appearance inshark development, and by their disulfide bond pattern [Diaz M,Stanfield R L, Greenberg A S, Flajnik, M F, Immunogenetics, 54, 501(2002); Nuttall S D, Krishnan U V, Doughty L, Pearson K, Ryan M T,Hoogenraad N J, Hattarki M, Carmichael J A, Irving R A, Hudson P J, Eur.J. Biochem. 270, 3543 (2003)].

The natural hetero-tetrameric structure of antibodies exists in humansand most animals. The heavy-chain only dimer structure is considerednatural characteristic of camelids and sharks [Holliger P, Hudson P J,Nature Biotechnology, 23, 1126 (2005)]. These antibodies are relativelysimple molecules but with unique characteristics. Since the variableantigen binding (Vab) site binds its antigen only through theheavy-chain, these antibodies are also known as single-domain antibodies(sd-Abs). Their size is about one-half the size of traditionaltetrameric antibodies, hence a lower molecular weight (˜80 KDa to 90KDa), with similar antigen binding affinity, but with water solubility100- to 1000-fold higher than conventional antibodies.

Another characteristic of heavy-chain antibodies derived from sharks andcamelids is that they have very high thermal stability compared to theconventional mAbs. For example, camelid antibodies can maintain theirantigen binding ability even at 90° C. [Biochim. Biophys. Acta., 141, 7(1999)]. Furthermore, complementary determining region 3 (CDR3) ofcamelid Vab region is longer, comprising 16-21 amino acids, than theCDR3 of mouse VH region, comprising 9 amino acids [Protein Engineering,7, 1129 (1994)]. The larger length of CDR3 of camelid Vab region isresponsible for higher diversity of antibody repertoire of camelidantibodies compared to conventional antibodies.

In addition to being devoid of light chains, the camelid heavy-chainantibodies also lack the first domain of the constant region called CH1,though the shark antibodies do have a CH1 domain and two additionalconstant domains, CH4 and CH5 [Nature Biotech. 23, 1126 (2005)].Furthermore, the hinge regions (HRs) of camelid and shark antibodieshave an amino acid sequence different from that of normalheterotetrameric conventional antibodies [Muyldermans S, Reviews in Mol.Biotech., 74, 277 (2001)]. Without the light chain, these heavy-chainantibodies bind to their antigens by one single domain, the variableantigen-binding domain of the heavy-chain immunoglobulin, which isreferred to as Vab in this application (VHH in the literature), todistinguish it from the variable domain VH of the conventionalantibodies.

The single-domain Vab is surprisingly stable by itself, without havingto be attached to the heavy-chain. This smallest intact andindependently functional antigen-binding fragment Vab, with a molecularweight of ˜12-15 KDa, derived from a functional heavy-chain only fulllength IgG, is referred to as a “nanobody” In the literature[Muyldermans S, Reviews in Mol. Biotech., 74, 277 (2001)].

The genes encoding these full length single-domain heavy-chainantibodies and the antibody-antigen binding fragment Vab (camelid andshark) can be cloned in phage display vectors, and selection of antigenbinders by panning and expression of selected Vab in bacteria offer avery good alternative procedure to produce these antibodies on a largescale. Also, only one domain has to be cloned and expressed to producein vivo an intact, matured antigen-binding fragment.

There are structural differences between the variable regions of singledomain antibodies and conventional antibodies. Conventional antibodieshave three constant domains while camelid has two and shark has fiveconstant domains. The largest structural difference is, however, foundbetween a VH (conventional antibodies) and Vab (heavy-chain onlyantibodies of camelid and shark) (see below) at the hypervariableregions. Camelid Vab and shark V-NAR domains each display surface loopswhich are larger than for conventional murine and human IgGs, and areable to penetrate cavities in target antigens, such as enzyme activesites and canyons in viral and infectious disease biomarkers [Proc.Natl. Acad. Sci. USA., 101, 12444 (2004); Proteins, 55, 187 (2005)]. Inhuman and mouse the VH loops are folded in a limited number of canonicalstructures. In contrast, the antigen binding loop of Vab possess manydeviations of these canonical structures that specifically bind intosuch active sites, therefore, represent powerful tool to modulatebiological activities [K. Decanniere et al., Structure, 7, 361 (2000)].The high incidence of amino acid insertions or deletions, in or adjacentto first and second antigen-binding loops of Vab will undoubtedlydiversify, even further, the possible antigen-binding loopconformations.

Though there are structural differences between camelid and shark parentheavy-chain antibodies, the antigen-antibody binding domains, Vab andV-NAR, respectively, are similar. The chemical and/or protease digestionof camelid and shark antibodies results in Vab and V-NAR domains, withsimilar binding affinities to the target antigens [Nature Biotech., 23,1126 (2005)].

Other structural differences are due to the hydrophilic amino acidresidues which are scattered throughout the primary structure of Vabdomain. These amino acid substitutions are, for example, L45R, L45C,V37Y, G44E, and W47G. Therefore, the solubility of Vab is much higherthan the Fab fragment of conventional mouse and human antibodies.

Another characteristic feature of the structure of camelid Vab and sharkV-NAR is that it often contains a cysteine residue in the CDR3 inaddition to cysteines that normally exist at positions 22 and 92 of thevariable region. The cysteine residues in CDR3 form S—S bonds with othercysteines in the vicinity of CDR1 or CDR2 [Protein Engineering, 7, 1129(1994)]. CDR1 and CDR2 are determined by the germline V gene. They playimportant roles together with CDR3 in antigenic binding [NatureStructural Biol., 9, 803 (1996); J. Mol. Biol., 311, 123 (2001)]. Likecamelid CDR3, shark also has elongated CDR3 regions comprising of 16-27amino acids residues [Eur. J. Immunol., 35, 936 (2005)].

The germlines of dromedaries and llamas are classified according to thelength of CDR2 and cysteine positions in the V region [Nguyen et al.,EMBO J, 19, 921 (2000); Harmsen et al., Mol. Immun., 37, 579 (2000)].

Immunization of camelids with enzymes generates heavy-chain antibodies(HCAb) significant proportions of which are known to act as competitiveenzyme inhibitors that interact with the cavity of the active site [M.Lauwereys et al., EMBO, J. 17, 3512 (1998)]. In contrast, theconventional antibodies that are competitive enzyme inhibitors cannotbind into large cavities on the antigen surface. Camelid antibodies,therefore, recognize unique epitopes that are out of reach forconventional antibodies.

Production of inhibitory recombinant Vab that bind specifically intocavities on the surface of variety of enzymes, namely, lysozyme,carbonic anhydrase, alfa-amylase, and beta-lactamase has been achieved[M. Lauwereys, et al., EMBO, J. 17, 3512 (1998)]. Hepatitis C proteaseinhibitor from the camelised human VH has been isolated against an 11amino. Eng. acid sequence of the viral protease [F. Martin et al., Prot,10, 607 (1997)].

SUMMARY OF THE INVENTION A. Single-Domain Heavy-Chain Only Antibodiesand Peptide Compositions Thereof

The present invention is intended to meet a large unmet medical need fornon-invasive diagnosis and treatment of diseases of the central nervoussystem (CNS). In a first aspect, the present invention teaches peptidecompositions of camelid and/or shark single-domain heavy-chain onlyantibodies and their synthetic peptide composition analogs for breachingthe blood-brain barrier (BBB) and cell membranes for diagnosing and/ortreating human diseases, including but not limited to, diseases of thecentral nervous system (CNS) and cancer. FIG. 2 presents the peptidecomposition structures.

This invention covers single-domain antibodies, and their syntheticpeptide composition analogs cross the BBB into the central nervoussystem and pharmaceutically acceptable formulations of the same. Theirgeneral configuration is shown by structures 1 and 2 in FIG. 2. Eachstructure in FIG. 2 contains one or two Vab domains, and each Vab domainis derived from an antigen-sdAb of camelid (Vab), shark (V-NAR), orcombinations thereof.

B. Production of Single-Domain Antibodies

In a second aspect, the invention is also how single-domain antibodiesof structures 1 and 2 from FIG. 2 can be produced from the serum ofcamelids or sharks immunized by an immunogen involved in a CNSdisease-causing process. These immunogens can be produced by chemicalsynthesis and conjugation to BSA or KLH for immunization, or throughrecombinant DNA technology.

1. Cloning the Single Heavy-Chain from Single-Domain Antibodies

The invention in which the single heavy-chain from a single-domainantibody can be produced by the techniques of the recombinant DNAtechnology involving isolation of peripheral blood lymphocytes,extracting total mRNA, reverse transcription to cDNA encoding thepeptide composition 2a (FIG. 2, Structure 2, Tables 2-6, variant: R1=1,R2=4, R3=2, L1=1, L2=1, R4=1, R5=1, X=1, Y=1), amplification of the cDNAby PCR, cloning in an appropriate vector, recovering and sequencing thecloned cDNA, cloning the sequenced fragment in a phase vector,transforming the host E. coli cells, and purifying the expressedprotein, followed by ELISA and Western blot analysis.

The invention in which the PCR primers are represented by SEQ ID NO: 1and SEQ ID NO: 2.

5′-------------------------------------------------3′  (SEQ ID NO: 1)CAG GTT CAG CTT GTT GCT TCT GGT  (SEQ ID NO: 2)TTT ACC AGG AGA AAG AGA AAG 

The invention in which a second round of PCR is done with primerscontaining built in restriction sites such as Xho and Not1 compatiblewith commercially available cloning vectors such as SEQ ID NO: 3 and SEQID NO: 4.

5′-------------------------------------------------------------3′ (SEQ ID NO: 3) CTCGAG-CAG GTT CAG CTT GTT GCT TCT GGT  (SEQ ID NO: 4)GCGGCCGC- TTT ACC AGG AGA AAG AGA AAG 

The invention in which cDNA sequence encoding the single heavy-chain ofcamelid antibody 2a is represented by the Camelid heavy-chain ofsingle-domain antibody in SEQ ID NO: 5. The lower-case letters arenucleotides at variable positions.

5′-----------Variable Antigen-Binding Domain (Vab)------------------------3′ (SEQ ID NO: 5)(1) CAG GTT CAG CTT GTT GCT TCT GGT GGT GGC TCT GTT CAG GCT GGT GGT TCT CTT CGT CTT (61) TCT TGT GCT GCT TCT GGT TAT ACT TTT TCT TCT TAT CCT ATG ggt tgg TAT CGT ggt gct (121) CCT ggt AAA GAA tgt GAA CTT TCT gct CGT ATT TTT TCT GAT ggt TCT gct AAT TAT gct (181) GAT TCT GTT AAA ggt CGT TTT act ATT TCT CGT GAT AAT gct gct AAT act gct TAT CTT (241) ggt ATG GAT TCT CTT AAA CCT GAA GAT act gct GTT TAT TAT tgt gct gct ggt CCT ggt (301) TCT ggt AAA CTT GTT GTT gct ggt CGT act tgt TAT ggt CCT AAT TAT TGG ggt ggc ggt  (361) act CAG GTT act GTT TCT TCT (381) Hinge-Region (HR) (382) GAA CCT AAA ATT CCT CAG CCT CAG CCT AAA CCT CAG CCT CAG CCT CAG CCT CAG CCT AAA (442) CCT CAG CCT AAA CCT GAA CCT GAA tgt act tgt CCT AAA tgc CCT (486)  Constant Domain-2 (CH2) (487) gct CCT CCT GTT gcc ggc CCT TCT GTT TTT CTT TTT CCT CCT AAA CCT AAA GAT act CTT (547) ATG ATT TCT CGT act CCT GAA GTT act tgt GTT GTT GTT GAT GTT TCT cat GAA GAT CCT (607) GAA GTT CAG TTT AT TGG TAT GTT GAT ggt GTT GAA GTT cat AT gcc AAA act AAA CCT (667) CGT GAA GAA CAG TTT AAT TCT act TTT CGT GTT GTT TCT GTT CTT act GTT GTT cat CAG (727) GAT TGG CTT AAT ggt AAA GAA TAT AAA tgt AAA GTT TCT AAT AAA ggt CTT CCT gct CCT (787) ATT GAA AAA act ATT TCT AAA act AAA (813) Constant Domain-3 (CH3) (814) ggc CAG CCT CGT GAA CCT CAG GTT TAT act CTT CCT CCT TCT CGT GAA GAA ATG act AAA (874) AAT CAG GTT TCT CTT act tgt CTT GTT AAA ggt TTT TAT CCT TCT GAT ATT GTT GAA TGG (934) GAA TCT AT ggc CAG CCT GAA AAT AAT TAT AAA act act CCT CCT ATG CTT GAT TCT GAT (994) ggt TCT TTT TTT CTT TAT TCT AAA CTT act GTT GAT AAA TCT CGT TgG CAG CAG ggt AAT (1054) GTT TTT TCT tgt TCT GTT ATG cat GAA gct CTT cat AAT cat TAT act CAG AAA TCT CTT  (1114) TCT CTT TCT CCT ggt AAA (1131) 

More specifically, the invention in which the immunogen is derived fromamyloid-peptide-42 (Aβ-42) (SEQ ID: 6), a constituent of amyloid-plaquefound in the brain of Alzheimer's patients.

SEQ ID NO: 6 D A E F R H D S G Y E V H H Q K L V F F A E D V GS N K G A I I G L M V G G V V I A 

Still more specifically, the immunogen is one of the following peptidesequences derived from the Aβ1-42 peptide:

(SEQ ID NO: 7) D A E F H R D S G Y E V H H Q K L V F F A E D V GS N K G A I I G L M C  (SEQ ID NO: 8) C D A E F H R D S G Y E V H H Q K (SEQ ID NO: 9) C E D V G S N K G A I I G L M (SEQ ID NO: 10)D A E F H R D S G Y E V H H Q K 

2. Target Biomarkers for Single-Domain Antibodies

The invention, wherein the camelid, shark, or a combination thereof,single-domain antibody configuration 1 and 2 in FIG. 2 are generatedfrom camelids and/or sharks immunized with immunogen(s) selected fromthe group of proteins or their metabolic products implicated inneurodegenerative diseases, including but not limited to, proteinsdescribed below.

Alzheimer's Disease (AD) Biomarkers.

Aβ, Tau protein, Tau-kinases (tyrosine kinase Fyn, glycogen synthasekinase 3 (GSK-3), cyclin-dependent protein-kinase-5, casein kinase-1,protein kinase-A, protein kinase-C, calcium and calmodulin-dependentprotein-kinase-II, MAPK), ApoE4, beta-secretase, gamma-secretase,translocase of the outer membrane (TOM), TDP43, ApoE4, c-terminal ofApoE4, GSK-3, acetylcholinesterase, NMDA (N-methyl-D-aspartate)receptor, APP (amyloid precursor protein), or ALZAS.

Parkinson's Disease (PD) Biomarkers.

Alpha-synuclein (Natural and mutant), LRRK2 (Natural and mutant),Parkin, DJ-1, Pink1, or Synphilin.

Multiple Sclerosis (MS) Biomarkers.

VIP (vasoactive intestinal peptide), PACAP (pituitary adenylatecyclase-activating peptide), Factor H, NF-L (neurofilament-light chain),NF-H (neurofilament-heavy chain), Tau, Aβ-42, Antitubulin, NSE(neuron-specific enolase), Apo-E, GAP-43 (growth-associated protein 43),24S—OH-chol (24S-hydroxycholesterol), Protein 14-3-3, sVCAM(solublevascular cell adhesion molecule), or sPECAM (soluble plateletendothelial cell adhesion molecule).

Glioblastoma Biomarkers.

EGFR, HER2, PDGF (platelet-derived growth factor), FGFR (fibroblastgrowth factor receptor), STATs (signal transducers and activators oftranscription), GDNF (glial cell-line derived neurotrophic factor), mTOR(mammalian target of rapamycin), VEGF (vascular endothelial growthfactor), TGF-beta (transforming growth factor beta), P13K, Ras, Raf,MAPK, AKT (aka: Protein Kinase B), MAPK, TIMP1, CD133, SPP1 (secretedphosphoprotein 1), TP53, PTEN, TMS1, IDH1, NF1, or IL-10.

Huntington's Disease Biomarkers.

H2aFY, mutant HTT, 8-Hydroxy-2-deoxy-guanosine, Copper-Zn SuperoxideDismutase, A2a receptor, transglutaminase, or poly-glutamine.

In preferred embodiments, the target biomarker to which the inventivepolypeptides specifically bind is an extracellular protein within theCNS or is a membrane-bound protein for which the antigenic epitope isaccessible by the polypeptide from the extracellular space (e.g., anextracellular domain of a membrane-bound protein).

3. Camelid Single-Chain Aβ-sdAb Sequence

The present inventions are based upon the discovery that a single domainheavy chain-only antibody derived from camelids and/or shark retainsantigen binding activity and is capable of crossing theblood-brain-barrier of mammals. In its simpliest form, the presentinventions are based on compositions and methods for using native ormodified camelid and/or shark single domain polypeptides derived from,or based on, the native heavy chain-only antibodies. For example, thenative single domain polypeptides of camelid heavy chain-only antibodieshave the general form of: Vab-hinge region (HR)-CH2-CH3, as illustratedin FIG. 16. In some embodiments, the Vab domain may be all or a portionof the native Vab domain provided that the Vab domain retains itsantigen binding capacity. In other embodiments, the hinge region may beall or a portion of the native hinge region and/or may be a non-peptidechemical linker as described herein. The CH domains may be the CHdomains native to the species of the Vab domain, or fragments thereof,or may be derived from another species, or fragments thereof. Forexample, a single domain heavy chain polypeptide may have a camelid Vabdomain and HR, one or more CH domains derived from a human IgG and/orone or more CH domains derived from shark IgG either with or without oneor more of the camelid CH domains. Optionally, the single domain heavychain only polypeptide may further comprise a peptide and/or non-peptidelinker and another moiety (e.g., ligand or a second single domain heavychain polypeptide, or CH domain from another species such as camelid,shark, or human.

The invention in which the amino-acid sequence of the single polypeptideheavy-chain is represented by the amino acid sequence of peptidecomposition 2a (SEQ ID NO: 11, the amino acid sequence of a camelidsingle-domain heavy-chain only antibody), which is a single chain frompeptide composition 1a (FIG. 2, structure 1, R=1), a camelid antibody.

Amino Acid Sequence of Camelid Single-Domain Heavy-Chain Only Antibody(Peptide Composition 2a, SEQ ID: 11)

Variable Antigen-Binding Domain (Vab) (1) QVQLVASGGG SVQAGGSLRL SCAASGYTFS SYPMGWYRGA PGKECELSAR IFSDGSANYA DSVKGRFTIS RDNAANTAYL GMDSLKPEDT ADYYCAAGPG SGKLVVAGRT CYGPNYWGGG  TQVTVSS (127) Hinge-Region (HR)  (128) EPK IPQPQPKPQP QPQPQPKPQP KPEPECTCPK  CP (162) Constant Region 2  (163) APPVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTK (272)  Constant Region 3 (273) G QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (378) 

C. Peptide Compositions of Structure 1 in FIG. 2

The invention in which variable antigen-binding domains of heavy-chainsingle-domain shark and/or camelid antibodies are linked togetherthrough to a constant domain CH2 of human IgG, camelid IgG, or sharkIgNAR which is linked to a constant domain CH3 of human IgG, camelidIgG, or shark IgNAR to form bivalent Vab domains of single-domainheavy-chain only antibody of the general structure of structure 1 inFIG. 2. Exemplary variants of the “R” group in structure 1 from FIG. 2are listed in Table 1.

TABLE 1 Variants of R from Structure 1 in FIG. 2 R Variant Description 1H 2 a detectable label 3 a short-lived radioisotope including, but notlimited to, as ¹²³I, ¹²⁴I, ⁷⁷Br, ⁶⁷Ga, ⁹⁷Ru, ⁹⁹Tc, ¹¹¹In or ⁸⁹Zrintroduced either using a reagent such as ¹²⁴I- Bolton Hunker or¹²⁴I-SIB or a metal chelator 4 a long-lived radionisotope including, butnot limited to, as ¹³¹I, ²¹¹At, ³²P, ⁴⁷Sc, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴⁰la,¹¹¹Ag, ⁹⁰Y, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁹⁹Au for therapeutic applications, with orwithout a labeling entity, such as a bifunctional reagent (Bolton Hunteror SIB reagent) or a bifunctional chelating agent between thepolypeptide and the radionuclide 5 NH—CO—C₆H₄-Iodine-124, 125, 111, 113,112, or 131 6 a fluorophore including, but no limited to, FITC and otherfluorescein derivatives, Texas-Red, rhodamine, Cy3, Cy5, thioflavindyes, AlexaFluor, 1,4-Bis(4′-aminostyryl)-2-dimethoxy-benzene (BDB;Formula 1a), [5′-(4-methoxyphenyl)[2,2′-bithiophen]-5yl]methylene]-propanedinitrile (Formula1b) [5′-(4-methoxyphenyl)[2,2′-bithiopehen]-5yl]-aldehyde (Formula 1c),or fluorophore analogs thereof 7 a therapeutic agent, toxin, hormone, orpeptide 8 a protein, such as an enzyme 9 an antibody, the Fc region ofIgGs, sd-insulin-Ab 1 (Structure 1 in FIG. 2) sd-insulin-Ab 2 (Structure2 in FIG. 2), sd-transferrin-Ab 1, or sd- transferrin-Ab 2 10 biotin,digoxigenin, avidin, or streptavidin 11 fused or covalently bound to aprotein that recognizes or binds to the receptors on endothelial cellsthat form the BBB, including, but not limited to, insulin, transferrin,Apo-B, Apo-E, such as Apo-E4, Apo-E Receptor Binding Fragment, FC5,FC44, substrate for RAGE (receptor for advanced glycation end products),substrate for SR (macrophage scavenger receptor), substrate for AR(adenosine receptor), RAP (receptor-associated protein), IL17, IL22, orprotein analogs thereof 12 nucleic acids including, but not limited to,a gene, vector, si-RNA, or micro- RNA 13 covalently bound tobiodegradable nanoparticles, such as polyalkylcyanoacrylatenanoparticles (PACA-NPs), wherein the PACA nanoparticles are synthesizedfrom a substituted surfactant, wherein the surfactant is dextran,polyethylene glycol, heparin, and derivatives thereof; wherein thesubstitution is that of an amino group, thiol group, aldehydic group,—CH2COOH group, with or without appropriate protection, for subsequentcovalent conjugation of the said polypeptide

Variants in Table 1 include mutants of the peptides, proteins, andnucleic acid sequences. The variants may include a bifunctional linkingmoiety including, but not limited to, peptides, such asglycyl-tyrosyl-glycyl-glycyl-arginine (SEQ ID NO: 12);tyramine-cellobiose (Formula 2); Sulfo-SMCC; NHS—(CH₂—CH₂—O)n-Mal(wherein n=1-100, NHS stands for N-hydroxysuccinimide, and Mal standsfor maleimido group); Succinimidyl-3 (4-hydroxyphenyl)-propionate;(3-(4-hydroxyphenyl) propionyl-carbonylhydrazide; EDTA(ethylenedinitrilotetraaceticacid); DTPA (diethylenetriaminepentaaceticacid) and DTPA analogs (Formula 3); NTA (N,N′,N″-triacetic acid);chelating agents such as desferroxamine (DFA) and bifunctional linkeranalogs thereof. EDTA derivatives include1-(p-bromoacetamidophenyl)-EDTA, 1-(p-benzenediazonium)-EDTA,1-(p-bromoacetamindophenyl)-EDTA, 1-(p-isothiocyanatobenzyl)-EDTA, or1-(p-succinimidyl-benzyl)-EDTA.

D. Peptide Compositions of Structure 2 in FIG. 2

The invention in which one or more variable antigen-binding domains(from camelid Vab, shark V-NAR, or a combination thereof) of heavy-chainpolypeptides are chemically or enzymatically linked together through ahinge region (HR), a non-peptidyl linker (such as a PEG linker), orcombination thereof, to a constant domain CH1, CH2 or CH3 of human IgGCH2 or CH3 camelid IgG, or CH1, CH2, CH3, CH4, or CH5 shark IgNAR toform a bivalent Vab domains of single-domain heavy-chain only antibodiesof the general structure of structure 2 in FIG. 2. Exemplary variants ofthe “R1,” “R2,” “R3,” “L1,” “L2,” “R4,” “R5,” “X,” and “Y” groups instructure 1 from FIG. 2 are listed in Tables 2-6.

TABLE 2 Variants of R1 or R2 from Structure 2 in FIG. 2 R1 or R2 VariantDescription 1 All or a part of a variable antigen-binding domain of asingle-domain antibody (Vab-sdAb) for an antigen, where the Vab isderived from camelid Vab, shark V-NAR, or a combination thereof 2 aConstant domain 1 (CH1) of Hu-IgG 3 a Constant domain 2 (CH2) of Hu-IgG4 a Constant domain 3 (CH3) of Hu-IgG 6 a Constant domain 2 (CH2) ofcamelid-IgG 7 a Constant domain 3 (CH3) of camelid-IgG 8 a Constantdomain 1 (CH1) of shark-IgNAR 9 a Constant domain 2 (CH2) of shark-IgNAR10 a Constant domain 3 (CH3) of shark-IgNAR 11 a Constant domain 4 (CH4)of shark-IgNAR 12 a Constant domain 5 (CH5) of shark-IgNAR

Variants include mutants of the constant domains. At least R1 or R2=1.

TABLE 3 Variants of R3 from Structure 2 in FIG. 2 R3 Variant Description1 a Constant domain 1 (CH1) of Hu-IgG 2 a Constant domain 2 (CH2) ofHu-IgG 3 a Constant domain 3 (CH3) of Hu-IgG 4 a Constant domain 2 (CH2)of camelid-IgG 5 a Constant domain 3 (CH3) of camelid-IgG 6 a Constantdomain 1 (CH1) of shark-IgNAR 7 a Constant domain 2 (CH2) of shark-IgNAR8 a Constant domain 3 (CH3) of shark-IgNAR 9 a Constant domain 4 (CH4)of shark-IgNAR 10 a Constant domain 5 (CH5) of shark-IgNAR

Variants include mutants of the constant domains.

TABLE 4 Variants of L1 or L2 from Structure 2 in FIG. 2 L1 or L2 VariantDescription 1 a hinge-region of a sdAb comprising of up to 35 aminoacids, wherein the amino acid sequence isEPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP (SEQ ID NO: 13) or a portion thereof2 a linker up to 20 nm long in length, wherein the linker is comprisedof polyethylene glycol (CH₂—CH₂—O))_(n) and n = 5-70 3 a linkercomprising from a group consisting of NHS-(CH₂—CH₂—O)n-Mal, wherein n =1-100, NHS stands for N-hydroxysuccinimide, and Mal stands for maleimidogroup; Succinimidyl-3 (4-hydroxyphenyl)-propionate; (3-(4-hydroxyphenyl)propionyl-carbonylhydrazide; EDTA (ethylenedinitrilotetraaceticacid),DTPA (diethylenetriaminepentaacetic acid), or NTA (N,N′,N″-triaceticacid) 4 alkoxy, alkyl, peptidyl, nucleic acid, unsaturated aliphaticchains or combinations thereof

Variants include mutants of the constant domains.

TABLE 5 Variants of R4 or R5 from Structure 2 in FIG. 2 R4 or R5 VariantDescription 1 H 2 a detectable label 3 a short-lived radioisotopeincluding, but not limited to, as ¹²³I, ¹²⁴I, ⁷⁷Br, ⁶⁷Ga, ⁹⁷Ru, ⁹⁹Tc,¹¹¹In or ⁸⁹Zr introduced either using a reagent such as ¹²⁴I- BoltonHunker or ¹²⁴I-SIB or a metal chelator 4 a long-lived radionisotopeincluding, but not limited to, as ¹³¹I, ²¹¹At, ³²P, ⁴⁷Sc, ⁶⁷Cu, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴⁰la, ¹¹¹Ag, ⁹⁰Y, ¹⁰⁵Rh, ¹⁰⁹Pd, or ¹⁹⁹Au for therapeuticapplications, with or without a labeling entity such as a bifunctionalreagent (Bolton Hunter or SIB reagent) or a bifunctional chelating agentbetween the polypeptide and the radionuclide 5 NH—CO—C₆H₄-Iodine-124,125, 111, 113, 112, or 131 6 a fluorophore including, but no limited to,FITC and other fluorescein derivatives, Texas-Red, rhodamine, Cy3, Cy5,thioflavin dyes, AlexaFluor, 1,4-Bis(4′-aminostyryl)-2-dimethoxy-benzene(BDB; Formula 1a),[5′-(4-methoxyphenyl)[2,2′-bithiophen]-5yl]methylene]- propanedinitrile(Formula 1b) [5′-(4-methoxyphenyl)[2,2′-bithiopehen]- 5yl]-aldehyde(Formula 1c), or fluorophore analogs thereof 7 a therapeutic agent,toxin, hormone, or peptide 8 a protein, such as an enzyme 9 an antibody,the Fc region of IgGs, sd-insulin-Ab 1 (Structure 1 in FIG. 2)sd-insulin-Ab 2 (Structure 2 in FIG. 2), sd-transferrin-Ab 1, or sd-transferrin-Ab 2 10 biotin, digoxegenin, avidin, streptavidin 11 fusedor covalently bound to a protein that recognizes or binds to thereceptors on endothelial cells that form the BBB, including, but notlimited to, insulin, transferrin, Apo-B, Apo-E, such as Apo-E4, Apo-EReceptor Binding Fragment, FC5, FC44, substrate for RAGE (receptor foradvanced glycation end products), substrate for SR (macrophage scavengerreceptor), substrate for AR (adenosine receptor), RAP(receptor-associated protein), IL17, IL22, or protein analogs thereof 12nucleic acids including, but not limited to, a gene, vector, si-RNA, ormicro-RNA 13 covalently bound to biodegradable nanoparticles, such aspolyalkylcyanoacrylate nanoparticles (PACA-NPs), wherein the PACAnanoparticles are synthesized from a substituted surfactant, wherein thesurfactant is dextran, polyethylene glycol, heparin, and derivativesthereof; wherein the substitution is that of an amino group, thiolgroup, aldehydic group, —CH2COOH group, with or without appropriateprotection, for subsequent covalent conjugation of the said polypeptide

Variants include mutants of the peptides, proteins, and nucleic acidsequences. The variants may include a bifunctional linking moietyincluding, but not limited to, peptides, such asglycyl-tyrosyl-glycyl-glycyl-arginine (SEQ ID NO: 12);tyramine-cellobiose (Formula 2); NHCO—(CH₂—CH₂—O)n (wherein n=1-100,5-75, 5-50, 5-25, or 10-20), Sulfo-SMCC; NHS—(CH₂—CH₂—O)n-Mal (whereinn=1-100, 5-75, 5-50, 5-25, or 10-20, NHS stands forN-hydroxysuccinimide, and Mal stands for maleimido group);Succinimidyl-3 (4-hydroxyphenyl)-propionate; (3-(4-hydroxyphenyl)propionyl-carbonylhydrazide; EDTA (ethylenedinitrilotetraaceticacid);DTPA (diethylenetriaminepentaacetic acid) and DTPA analogs (Formula 3);NTA (N,N′,N″-triacetic acid); chelating agents such as desferroxamine(DFA) and bifunctional linker analogs thereof. EDTA derivatives include1-(p-bromoacetamidophenyl)-EDTA, 1-(p-benzenediazonium)-EDTA,1-(p-bromoacetamindophenyl)-EDTA, 1-(p-isothiocyanatobenzyl)-EDTA, or1-(p-succinimidyl-benzyl)-EDTA.

TABLE 6 Variants of X or Y from Structure 2 in FIG. 2 X or Y VariantDescription 1 a bifunctional peptidyl, alkyl-, alkoxy-, aromatic,nucleotide linker or a combination thereof including any of theforegoing variants of L.

E. Peptide Nanoparticle Compositions of Structures 1 and 2 in FIG. 2

Any of the polypeptides of the invention, including the structures 1 or2 in FIG. 2 and the single domain polypeptides, may be covalently linkedto biodegradable nanoparticles, which are comprised of a surfactant,ploymerizable monomeric entity, wherein surfactant is carrying afunctionally active group such as amino group, aldehyde group, thiolgroup, —CH2-COOH group, succinic anhydride group or other group capableof forming a covalent bond between the nanoparticles and the peptidecompositions. The following examples exemplify these embodiments but theprinciples and disclosure may be applied to any of the polypeptidesdisclosed herein.

Structure 1 in FIG. 2 (at R) and structure 2 in FIG. 2 (at R4 and/or R5)may be conjugated to biodegradable nanoparticles. Wherein thenanoparticles are synthesized by the chemical reaction of biodegradablealkylcyanoacrylate with a substituted polymer such as dextran, heparin,polyethylene glycol, and the like; wherein the substitution is that of agroup, free or protected, capable of forming a covalent bond with thenative and/or modified single-domain antibody.

Wherein, in Formula 4, X=—NH, S, CHO, phosphate, thiophosphate, orphosphonate; and wherein, in Formula 4, Y=a peptide composition ofstructure 1 in FIG. 2 or a peptide composition 2 in FIG. 2, orcombinations and variants of structures 1 and 2.

F. Methods for Treatment

Any of the polypeptides of the invention, or derivatives thereof,disclosed herein may be used for treating neurologic diseases, includingneurodegenerative diseases in a mammal (e.g., a human). In oneembodiment, the polypeptides are administered to a subject in needthereof (e.g., a subject diagnosed as having or at risk of developing aneurologic disease) in an amount sufficient to treat or prevent thatneurologic disease, wherein the polypeptide specifically binds to atarget biomarker.

In a related method, the invention provides a method for reducing thebiological activity (e.g., amount) of a target biomarker in the centralnervous system (CNS) of a subject by administering a polypeptide of theinvention according to the methods disclosed herein.

Neurologic diseases amenable to treatment or prevention according tothese methods include, for example, Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, Huntington's disease, and cancers of thecentral nervous system such as glioblastoma.

The polypeptides are administered in an amount and duration sufficienttreat or prevent the neurological disease. For example, the polypeptidesmay be administered one or twice a day, or more frequently, about once aweek, or about once a month. The polypeptides may be administered as asingle (one-time) treatment or for a duration of a week, a month, twomonths, six months, one year, or more, or for the lifetime of thesubject. The polypeptides may be administered as a unitary dosage (e.g.,a dosage administered over a discrete duration of time) or by continuousinfusion. Administered doses of the polypeptide (i.e., “an amountsufficient”) may be about 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mgor more per day.

In preferred embodiments, the amount and/or biological activity of thetarget biomarker is reduced in the nervous system of the subjectfollowing administration of the polypeptide.

G. Methods for Diagnosis

Any of the polypeptides of the invention, or derivatives thereof,disclosed herein may be used for diagnosing a subject (e.g., a human) ashaving, or at risk of developing, a neurologic disease. One diagnosticmethod comprises (i) administering an inventive polypeptide comprising adetectable label to the subject, wherein the polypeptide binds to atarget biomarker, (ii) waiting for a time sufficient for the polypeptideto permeate the blood-brain-barrier and bind to the target biomarker, ifpresent, (iii) determining the presence or amount of detectable labelwithin the central nervous system (CNS), and (iv) identify the subjectas having, or at risk of developing, the neurologic disease when themeasured amount of the target biomarker is different from the measuredamount of the target biomarker in a subject or population of subjectsknown to be either disease-free or not at risk of developing thedisease. In some embodiments of this method, an increase or a decreasein the amount of target biomarker relative to the subject or populationof subjects known to be either disease-free or not at risk of developingthe disease indicates that the tested subject either has the disease oris at risk of developing the disease.

Another diagnostic method comprises (i) at a first time, administeringan inventive polypeptide comprising a detectable label to the subjectand obtaining a first measurement of the amount of the detectable labelwithin the CNS of the subject, wherein the polypeptide binds to a targetbiomarker; (ii) at a second time, administering an inventive polypeptidecomprising a detectable label to the subject and obtaining a firstmeasurement of the amount of the detectable label within the CNS of thesubject; and (iii) comparing the amount of detectable label within theCNS of the subject at the second time to the amount of detectable labelwithin the CNS of the subject at the first time, wherein a change in theamount of detectable label at the second time relative to the first timeindicates that the subject has, or is at risk of developing, theneurological condition. In some embodiments of this method, an increaseor a decrease in the amount of target biomarker at the second timerelative to the first time indicates that the tested subject either hasthe disease or is at risk of developing the disease. The duration oftime between the first measurement and the second measurement can be anytime that is convenient and appropriate for the disease underinvestigation. For example, the time may be at least about one day, oneweek, one month, two months, three months, six months, one year, twoyears, three years, five years, or more.

In another embodiment, the invention provides a method for labeling oneor more targets in the CNS of a vertebrate, such as a mammal or a human,using any of the foregoing methods, wherein the polypeptide comprises adetectable label.

In some embodiments of the foregoing diagnostic methods, the targetbiomarker is amyloid beta and the neurologic disease is Alzheimer'sdisease. In other embodiments, the target biomarker is LRRK2 and theneurologic disease is Parkinson's disease.

In any of the foregoing methods, it is contemplated that the presence oramount of detectable label detected or measured in the CNS of a subjectis indicative of the presence or amount target biomarker in the CNS ofthat subject.

H. Use of Single-Domain Heavy Chain Only Antibodies and Derivatives as aCNS Shuttle

Any of the polypeptides of the invention, or derivatives thereof,disclosed herein may be used to carry other diagnostic or therapeuticmolecules (“shuttled molecules”) into the CNS accordingly to the methodsset forth above. The shuttled molecules themselves may be capable ofcrossing the blood-brain-barrier, albeit on a limited basis, or may beimpermeate to the blood-brain-barrier. Thus, higher CNS levels of theshuttled molecules may be achieved by attaching such molecules to theinventive polypeptides according to the methods described herein. Insome embodiments, the shuttled molecule is a diagnostic agent (e.g., adetectable label) or a therapeutic agent for treating a cancer or otherneurologic disease. In other embodiments, the target biomarker to whichthe polypeptide binds is present on or near the target cells ofinterest. For example, when treating or diagnosing a glioblastoma,wherein the shuttled molecule is a chemotherapeutic agent or imagingagent, respectively, target biomarkers present on glioblastoma cells arepreferred. Likewise, when treating Alzheimer's disease or Parkinson'sdisease, target biomarkers on cholinergic and dompaninergic neurons,respectively, is preferend.

I. Specific Embodiments of Single Domain Heavy Chain Only Antibodies

In any of the foregoing methods and compositions, the followingembodiments of the polypeptides are particularly useful. In oneembodiment, the polypeptide comprising the formula: Vab-Z-Z′

-   -   wherein Vab comprises all or a portion of a variable        antigen-binding (Vab) domain of a camelid or shark single domain        heavy chain antibody,    -   wherein Z comprises all or a portion of a hinge region from an        IgG or a linker comprising —NHCO—,    -   wherein Z′ comprises a covalent bond or all or a portion of at        least one IgG CH domain, and    -   wherein the polypeptide is capable of specifically binding to a        target biomarker within the central nervous system (CNS) of the        subject;

The polypeptide optionally may further comprise a detectable labelincluding, for example, a radiolabel such as a positron-emittingradioisotope.

In one embodiment, the polypeptide contains only a single Vab of asingle domain heavy chain antibody lacking light chains. In furtherembodiments, this polypeptide contains a single camelid Vab covalentlyattached to a camelid CH2 and/or a camelid CH3 domain. The attachmentmay be through a hinge region and/or a chemical linker, as definedherein. In a related embodiment, the polypeptide comprises the formulaVab¹-Z¹-Z′-Z²-Vab², wherein Vab² independently comprises all or aportion of a variable antigen-binding (Vab) domain of a camelid or sharksingle domain heavy chain antibody and Z² independently comprises all ora portion of a hinge region from an IgG (e.g., camelid CH2 and/or acamelid CH3 domain).

In another embodiment, Z comprises a linker. Optionally, Z′ iscovalently attached to a second Vab-Z moiety such that the polypeptidecomprises the formula: Vab¹-Z¹-Z′—Z²-Vab², wherein Vab² independentlycomprises all or a portion of a variable antigen-binding (Vab) domain ofa camelid or shark single domain heavy chain antibody and Z²independently comprises all or a portion of a hinge region from an IgGand/or a linker.

In another embodiment, Z comprises all or a portion of a hinge regionfrom an IgG. Optionally, Z′ is covalently attached to a second Vab-Zmoiety such that the polypeptide comprises the formula:Vab¹-Z¹-Z′-Z²-Vab², wherein Vab² independently comprises all or aportion of a variable antigen-binding (Vab) domain of a camelid or sharksingle domain heavy chain antibody and Z² independently comprises all ora portion of a hinge region from an IgG and/or a linker.

In another embodiment, Z comprises all or a portion of a hinge regionfrom an IgG and a a linker. Optionally, Z′ is covalently attached to asecond Vab-Z moiety such that the polypeptide comprises the formula:Vab¹-Z¹-Z′-Z²-Vab², wherein Vab² independently comprises all or aportion of a variable antigen-binding (Vab) domain of a camelid or sharksingle domain heavy chain antibody and Z² independently comprises all ora portion of a hinge region from an IgG and/or a linker.

In any of the foregoing embodiments, Z′ may comprises all or a portionof a human IgG CH domain (e.g., CH1, CH2, and/or CH3), camelid CH domain(e.g., CH2 and/or CH3), and/or shark CH domain (e.g, CH1, CH2, CH3, CH4,and/or CH5).

In any of the foregoing embodiments, suitable chemical linkers include,for example, linkers which contain a PEG moiety such as —(CH₂CH₂—O)_(n)—and wherein n=2-100, 2-75, 2-50, or 2-24, (e.g., n is at least 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, or 24). In specific embodiments, thelinker comprises —NHCO—(CH₂CH₂—O)_(n)—, or —(C₄H₃NO₂)—S—(CH₂)₃-imidate-,or —CH(CH₂SH)—NHCO—, or—NHCO—(CH₂CH₂—O)_(n)—(C₄H₃NO₂)—S—(CH₂)₃-imidate-.

In any of the foregoing embodiments, the polypeptide optionally may belinked to a nanoparticle. In some embodiments, the nanoparticle has adiameter of about 10 nm to about 500 nm, is covalently linked to thenanoparticle via a linker comprising —NHCO— and related linkersdescribed herein, and/or comprises polybutylcyanoacrylate.

By “treatment,” when referring to the therapeutic methods of the presentinvention, is meant administration of a polypeptide in an amount andduration sufficient to ameliorate at least one symptom of the disease.Symptoms that may be ameliorated include clinical symptoms (e.g., tremorin PD or cognitive impairment in AD), anatomical (e.g., slowing orreversing neuronal loss), or biochemical (e.g., reducing the biologicalactivity the target biomarker).

By “prevention” is meant administering a polypeptide in an amount andduration sufficient to slow or halt a pathological change associatedwith the neurologic disease in a subject identified as being at risk ofdisease development (i.e., if the subject is asymptomatic and does nototherwise meet the criteria for a positive diagnosis) or diseaseprogression.

By “administration” is meant the delivery of a polypeptide of theinvention to the subject in need thereof in a manner designed toultimately result in the delivery of that polypeptide to the centralnervous system of the subject. Routes of administration may include oraland/or parenteral delivery. Parenteral delivery includes subcutaneous,intravenous, intramuscular, intrathecal, and intraventricular injection.

By “biological activity,” when referring to a target biomarker, is meantthe physiological activity normally associated with the target biomarkerprotein. For example, in the case of enzymes, the biological activityrefers to the normal catalytic activity of that enzyme. In the case ofstructural or other proteins lacking a catalytic activity, biologicalactivity refers to the normal functioning of that protein in a cell ofthe nervous system (e.g., the ability to polymerize with otherstructural proteins). Biological activity may be reduced by reducing theamount of the target biomarker protein and/or inhibiting the function ofthe target biomarker protein.

As used herein, “amyloid beta” may refer to the amyloid beta proteinitself and/or amyloid plaque including soluble, insoluble, anddiffusible amyloid plaques.

By “pharmaceutically acceptable formulation” is meant a formulationsuitable for administration to a subject (e.g., a mammal such as ahuman). Pharmaceutically acceptable formulations generally include apolypeptide of the invention and at least one compatible excipient.Co-formulations with additional therapeutic agents are alsocontemplated.

By “derived from,” when used in reference to the polypeptides of thepresent invention or individual domains thereof, is meant that thepolypeptide or domain has a primary amino acid sequence that issubstantially identical to a native polypeptide or domain.

By “substantially identical” is meant a polypeptide or nucleic acidsequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to a reference sequence (e.g., a naturally-occurring sequence)over the length of comparison which is at least 10, 15, 20, 25, 30, 40,50, or more monomeric units (e.g., nucleotides or amino acids) long. Insome embodiments, a substantially identical sequence is 100% identicalto the reference sequence. In other embodiments, with reference topolypeptides, the sequence is not identical to the reference sequencebut contains one, two, three, four, five, or more conservative aminoacid substitutions.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 is a diagram of the tight junctions in the endothelial cellmembrane that form the barrier between the blood and the brain.

FIG. 2 depicts generic blood-brain permeable peptide compositionstructures 1 and 2 where each structure contains at least one Vab domainand each Vab domain was derived from a camelid Vab, shark V-NAR, or acombination thereof, from one or more sdAbs for an antigen.

FIG. 3 is a photograph of affinity-purified Aβ-sdAb 1a on an SDS-PAGEgel.

FIG. 4 depicts the synthetic sequence of conjugating two peptides bynative chemical ligation.

FIG. 5 depicts the synthetic sequence of conjugating two peptides bymalemido and thiol groups.

FIG. 6 is a photograph of the ELISA results of Aβ-sdAb 1a and Pierce'sAβ-mAb binding affinity to the Aβ-42 peptide as measured by the OD450readings of the chromogenic yellow color generated by the reaction ofthe HRP secondary antibody with the TMB substrate.

FIG. 7 is a photograph of the immunostaining results ofimmunohistochemical (IHC) staining of paraffin embedded brain tissuesfrom the APP transgenic mouse (FIG. 7A left upper two frames) andAlzheimer's patient (FIG. 7A lower two frames); with and without primaryAβ-sdAb (FIG. 7A frames), and staining of the same tissues withThioflavin-S dye (FIG. 7B frames).

FIG. 8 depicts the BBB permeability tests for Aβ-sdAb 1a (FIG. 2,Structure. Table 1, Variant: R=1) and peptide composition a(single-chain of Aβ-sdAb 1a; FIG. 2, Structure 2 Tables 2-6, variant:R1=1, R2=4, R3=2, L1=1, L2=1, R4=1, R5=1, X=1, Y=1) in mice.

FIG. 9 is photographs of the immunostaining results from blood-brainbarrier (BBB) permeability studies of peptide composition 2a compared toAβ-Mouse-IgG in the live APP transgenic mice injected in their tailswith peptide composition 2a or AD-Mouse-IgG.

FIG. 10 is a graph of the serum retention time of the peptidecomposition 2a in Aβ-mice.

FIG. 11 depicts the generic synthetic sequence of synthesizing maleimidoderivatized peptide composition 3 from peptide composition 2 (structure2 in FIG. 2) and synthesizing thiolated dextran-PBCA-nanoparticles 6from dextran 4.

FIG. 12 depicts the generic conjugation, after the sequence in FIG. 11,of maleimido-peptide composition 3 to thiol-functionalizeddextran-coated PBCA-nanoparticles 6, forming covalently-coated peptidecomposition 2-nanoparticle 7.

FIG. 13 depicts the generic synthetic sequence of synthesizing maleimidoderivitized-sdAb8 from sd-Ab 1 (structure 1 in FIG. 2) and synthesizingthiolated dextran-PBCA-nanoparticles 6 from dextran 4.

FIG. 14 depicts the generic conjugation, after the sequence in FIG. 13,of maleimido-sdAb 8 to thiol-functionalized dextran-coatedPBCA-nanoparticles 6, forming covalently-coated sdAb-nanoparticle 2.

FIG. 15 is a structure of covalently-coated sdAb-nanoparticle 9(structure 1 in FIG. 2) that is comprised of a maleimodio linker to formcovalently-coated sdAb-nanoparticle 2 conjugated to anamino-dextran-coated-PBCA-nanoparticle 6.

FIG. 16 illustrates the structures of a native camelid heavy chain-onlyantibody (left) which is a dimer formed by intermolecular bonding of twomonomeric units (right), wherein R═H. Each monomer comprises thefollowing structure: Vab-hinge region (HR)-CH2-CH3

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following detailed description, reference is made to theaccompanying drawings, which form a part thereof. The illustrativeembodiments described in the detailed description, drawings and claimsare not meant to be limiting. Other embodiments may be utilized, andother changes may be made, without departing from the spirit or scope ofthe subject matter presented here. The present technology is alsoillustrated by the examples herein, which should not be construed aslimiting in any way.

A. ISOLATION OF Aβ-SINGLE-DOMAIN ANTIBODY 1A (Aβ-SDAB 1A, STRUCTURE 1 INFIG. 2)

1. Immunization of Camelids with Aβ1-35 Peptide

All animals (llamas) were treated following NIH guidelines. First, theanimals were given a complete physical examination by our veterinarian,Dr. Linda Byer, who also drew some pre-immunization blood. Immunizationwas then started with Aβ₁₋₃₅ synthetic peptide (200 ug) in Gerbu PharmaAdjuvant (1 ml). One month after the initial priming injection, sixbiweekly boosters were administered at 200 ug/injection. After thefourth booster, about 20 ml blood was drawn and serum examined forantibody titer with antigen coated 96-well ELISA plate. Afterimmunization, ˜200 ml blood was drawn from the animal. Half of the bloodwas used to isolate single-domain Aβ-antibody (polyclonal) with themethods below in Sections A.2-4. The second half of the blood was usedto isolate peripheral blood lymphocytes (PBLs) to prepare total RNAfollowed by its reverse transcription to cDNA, which was then ligatedinto the phage vector to generate phage-display cDNA library (SectionD).

2. Crude Isolation of Aβ-sdAb 1a in from Camelid Serum

After immunization, 100 ml from each blood sample drawn was processed tofractionate sdAbs (MW: ˜90 KDa) from the classical antibodies (MW: ˜160KDa). Briefly, the serum (˜50 ml) was concentrated on anMillipore-Amicon Ultra-15 Concentrator, molecular weight cutoff 50 KDa,by spinning the device at 4000 g, until most of the low molecular weightspecies passed through the membrane. The thick viscous yellow retentate(˜25 ml) was extracted with chloroform (3×25 ml) to remove fattysubstances, which had contributed to the viscosity of the retentate. Theresulting crude product (2×10 ml) was size fractionated on Superdex-200(2.5 cm×100 cm) using 1×PBS as eluant. The fractions were monitored byreading OD₂₈₀ on a Beckman DU-640 Spectrophotometer. After examining thefractions on a 12% SDS-PAGE gel, the fractions whose products correspondto the molecular weight of ˜90 KDa were pooled, concentrated and theprotein concentration was measured by checking its OD₂₈₀.

3. Generation of an Affinity Column for Enrichment of Aβ-sdAb 1a

10 mg of immunogen Aβ1-35, dissolved in 5 ml of conjugation buffer, 0.1M NaHCO₃/0.15M NaCl, pH 8.5, was conjugated with cyanogen-bromideactivated Sepharose (2 gm), which had been washed with 200 ml ofice-cold 1 mM HCl. The reaction was allowed to proceed for 2 hours whilethe resin was allowed to gently rock on a rocker. After centrifugation,the supernatant of the reaction mixture was examined by its OD₂₈₀reading, which indicated that essentially all of the immunogen had beenconsumed. The resin was then washed with pH 8.5 conjugation buffer (3×20ml), and then blocked with 1 M Tris.HCl, pH 8.3 (10 ml), roomtemperature for 2 hours. After washing the resin with 0.1 M NaHCO₃/0.5MNaCl, pH 8.5, the resin was washed with 0.1 M sodium citrate (50 ml), pH2.8 and equilibrated with 20 mM sodium phosphate buffer, pH 7.0, beforeusing the resin for affinity purification.

4. Affinity Purification of Aβ-sdAb 1a

The crude mixture of sdAbs obtained after size fractionation onSuperdex-200, which was more than 98% free of full-length conventionalIgGs, was allowed to incubate with the affinity column in 1×PBS, at roomtemperature for one hour. After one hour, the unbound material wasallowed to drain through the column and the column washed with PBS untilall the unbound proteins had been washed off the column. The boundAβ-sdAb was eluted off the column with pH 2.8 buffer (0.1 M sodiumcitrate, 0.2 um filtered). The eluant was adjusted to pH 7.2 by adding 1M Tris.HCl, pH 9.0, and concentrated on Millipore-Amicon Ultra-15concentrators (30 KDa molecular weight cutoff). The retentate was bufferexchanged to 1×PBS and stored at −20° C. to obtain 1.65 mg of Aβ-sdAb 1a(FIG. 2, Structure 1, Table 1, Variant: R=1). Its protein concentrationwas determined using Pierce's BCA Protein Assay Kit. The SDS-PAGEanalysis of the affinity purified Aβ-sdAb 1a is in FIG. 3. About 10 ugof the Aβ-sdAb 1a after each step was electrophoressed on 12% SDS-PAGEgel after loading in SDS-loading buffer. The electrophoresis wasperformed at 100 volts for one hour, the gel was stained in 0.04%Coomassie Blue stain for 30 minutes at room temperature (RT). Coomassieblue stained SDS-PAGE (12%) protein gel of sequentially purified Aβ-sdAb1a, panel D: 1^(st) and 2^(nd) purifications were on Superdex-200;3^(rd) purification was done by affinity chromatography.

B. Synthesis of Single-Chain Aβ-SDAB 2A and Epitope Mapping of PeptideComposition 2A

1. Isolation of Single-Chain Aβ-sdAb 2a from Aβ-sdAb 1a

1.0 mg of Aβ-sd-Ab 1a was dissolved in 400 ul of pH 7.4 PBS. To thissolution was added 100 ul of 100 mM triethoxy carboxyl-phospine (TCEP)in PBS to obtain a final concentration of 20 mM. The reaction mixturewas incubated at 4° C. for 12-15 hours when gel electrophoresis (10%SDS-PAGE) showed a low molecular weight species with molecular weight of˜50 KDa. This product peptide composition 2a (single chain of Aβ-sdAb1a) was isolated by gel filtration and tested by Western and ELISA.

2. Epitope Mapping of Single-Chain Aβ-sdAb 2a

96-Well microplates (A1-A12 through G1-12 wells) were coated intriplicate with 600 ng per well of the following syntheticamyloid-peptide segments of Aβ1-42 peptide in Table 7.

TABLE 7 Synthetic amyloid-peptide segments of Aβ1-42 peptide Peptidesegment Amino acid positions 1  1-16 2  5-20 3  9-24 4 13-28 5 17-32 621-37 7 25-41 8 29-42

After coating the plate at 4° C. for 12 hours, the antigens werediscarded and the wells washed with deionized water (3×). The plate wasblocked with 1% BSA in 50 mM Tris/150 mM NaCl, pH 7.5 for one hour. Atthe end of one hour, single-chain Aβ-sdAb, 2a, 1.0 ug diluted to 2500 ulwith 1% BSA/Tris buffer was added to the top row (100 ul per well intriplicates). After serial dilution all the way to 1:320000 ul, theplate was incubated with gentle shaking at room-temperature for 2 hours.At the end of 2 hour incubation, the plate was washed three times, 250ul per well, with 0.05% tween-20/PBS. After washing, the wells wereincubated with 100 ul per well of goat-anti-llama-IgG-HRP conjugate(Bethyl Labs, Texas) 1.0 ug diluted to 10 ml of 1% BSA in PBS. After onehour incubation, the plate was washed with 0.05% Tween as above. Thewashed well were treated with 100 ul of TMB substrate and the plate readat 370 nm. The highest antibody titer was detected with the peptide 1-16amino acid long.

Subsequently, two synthetic peptide were synthesized: the 1-8 and 9-16peptides from the amyloid beta peptide and the above ELISA was againrepeated with the plate coated with 600 ng of each of the peptide intriplicates. This time the peptide of the 9-16 amino acids gave thehighest antibody titer, and no reaction took place with the sequence 1-8mer. The epitope is between 9 to 16 amino acids with the followingsequence: G Y E V H H Q K (SEQ ID NO: 14).

C. Synthesis of Peptide Composition Structure 2 in FIG. 2 Derivatives

1. Protease Digestion of Single-Chain Aβ-sdAb 2a to Obtain Aβ-Vab-HR(Aβ-Vab with L1 or L2 Linker Variant)

Generation of Sepharose-Endoproteinase Glu-C Conjugate.

Endoproteinase Glu-C (Worthington Biochemical Corporation), 4 mg, wasconjugated to 250 mg of CNBr-activated Sepaharose (GE Healthcare,catalogue #17-0430-1) in pH 8.5 0.1 M NaHCO₃/0.5M NaCl in 1×10 cm longspin fitted with a medium fritted disc, as described in Section A.3:Generation of an Affinity Column for Enrichmant of Aβ-sdAb 1a. Afterconjugation, any unbound Glu-C proteinase was removed by extensivewashing of Sepharose and the column was stored in 0.1% NaN₃/PBS untilused. The Sepharose had swollen to about an 0.8 ml volume.

Digestion of Single-Chain Aβ-sdAb 2a and Isolation of Aβ-Vab-HR.

Aβ-sdAb 2a (1 mg, ˜11 nmols) was dissolved in 1.0 ml of pH 7.5 0.1 MNaHCO₃ and added to the 0.8 ml of Sepharose-Glu-C conjugate. Thereaction mixture was gently rocked on a rocker for 4 hours and thecontents were collected by draining the column and washing it with 4 mlof the conjugation buffer, 0.1 M NaHCO₃, pH 7.5. The combinedflowthrough was passed through Aβ₁₋₃₅-affinity column generated inSection A.3. After washing off the unbound material, the bound Aβ-Vab-HR(HR=hinge region) from single-chain Aβ-sdAb 2a was eluted with pH 2.80.1 M sodium citrate and the product buffer exchanged to 1×PBS, pH 7.4.It was tested by ELISA.

2. Methods for Linking Aβ-Vab-HR to Antibody Constant Domains

General Method for Expression of Engineered Human Antibody ConstantDomains, CH1, CH2 and CH3.

Expression of engineered human constant domains CH1, CH2 and CH3 wasaccomplished by buying the commercially available plasmid, pFUSE-CHIg(Invitrogen: pFUSE-CHIg-hG1, pFUSE-CHIg-hG2, or pFUSE-CHIg-hG3), andusing them each for transformation of E. coli strain HB2151 cells. Thecultures were grown in SB media at 37° C. until an optical density of˜0.7 was obtained. Expression was then induced with 1 mM IPTG(isopropyl-1-thio-b-D-galactopyranoside) at 37° C. for 15-16 hours. Thebacterial cells were harvested and resuspended in a culture mediumcontaining 10% of 50 mM Tris.HCl, 450 mM NaCl, pH 8.0. Polymyxin Bsulfate (PMS) was added to the culture medium, 1:1000 volume of PMS:culture volume. After centrifuging the cell lysate at 15000 RPM for 45minutes at 4° C., the supernatant was purified by HiTrap Ni-NTA columnand tested for the respective expressed human constant domain bySDS-PAGE and Western blot.

General Method for Native Chemical Ligation of Aβ-Vab-HR to HumanConstant Domain.

For native chemical ligation (FIG. 4), an unprotectedpeptide-alpha-carboxy thioester (peptide 1) was reacted with a secondpeptide (peptide 2) containing an N-terminal cysteine residue to form anatural peptide linkage between Aβ-Vab-HR and constant domain CH1 or CH2or CH3. Aβ-Vab-HR can be modified to be peptide 1 or peptide 2. The CH1,CH2, or CH3 domain is then modified to be the recipricol peptide 2 orpeptide 1. After the reaction, the Aβ-Vab-PEG-Human CH1, CH2, or CH3 waspurified by size exclusion chromatography.

General Method for Maleimido-Thiol Conjugation Chemical Linkage ofAβ-Vab-HR to Human Constant Domain.

For the maleimido-thiol conjugation reaction (FIG. 5), a thiolatedpeptide 2 conjugates to a maleimido-derivativized peptide 1 to createaliphatic linker between Aβ-Vab-HR and a CH1, CH2, or CH3 domain.Aβ-Vab-HR can be modified to be peptide 1 or peptide 2. The CH, CH2, orCH3 domain is then modified to be the recipricol peptide 2 or peptide 1.Peptide 1 was converted into a maleimido peptide by reacting in with20-fold excess of commercial NHS-PEG-Mal (MW: 3000 Da) in pH 7.0 MOPSbuffer (0.1 M MOPS/0.15 M NaCl) for one hour at room temperature. Afterthe reaction, excess PEGreagent was removed by dialysis on Vivaspin-20column with a MWCO: 10 KDa. To generate compatible reacting group,peptide 2 was thiolated with commercial Traut's reagent to obtainthiolated peptide 2. 1.2 molar equivalent of thiolated peptide 2 whichwas reacted with maleimido derivatized peptide 1 at pH 6.8 at roomtemperature for 2 hours. After the reaction, the Aβ-Vab-PEG-Human CH,CH2, or CH3 was purified by size exclusion chromatography.

D. Phage-Display cDNA Library Generation of Aβ-SDAB 1

1. Cloning of cDNA Encoding the Aβ-sdAb 1a: mRNA Isolation and ReverseTranscription

The isolation of total RNA from peripheral blood lymphocytes (PBLs) from100 ml blood samples from immunized animals and subsequent reversetranscription to cDNA was done using commercial kits, such as PAXgeneBlood RNA Tubes and Blood RNA Kit system (Qiagen, Mississauga, ON).

2. PCR Amplification of cDNA and Construction of Expression Vector

Amplification of cDNA was done using PCR with primers SEQ ID NO:1 andSEQ ID NO:2. The second round of PCR amplification was done usingprimers with built-in restriction enzyme sites (SEQ ID NO: 3 and SEQ IDNO: 4) for insertion into pHEN4 phagemid, which was used to transformbacterial cells (WK6 E. coli). The clones were sequenced by the dideoxysequencing method. Sequences were then translated so that they can beassigned to well defined domains of the sdAb.

3. General Method for Expression and Purification of Aβ-sdAb 1

The bacterial cells containing the proper plasmids were grown, andexpression of the recombinant proteins induced with 1 mMisopropyl-β-D-thiogalactopyranoside (IPTG). The periplasmic proteinswere extracted by osmotic shock in the presence of protease inhibitors[(4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) and leupeptin)], andrecombinant protein purified by immobilized metal affinitychromatography (Ni-NTA Superflow, Qiagen). The MALDI-TOF massspectrometry of Aβ-sdAb 1a in sinapinic acid displayed a molecular ionat m/e 84873.5842. The molecular weight was validated by SDS-PAGE andWestern blot. The purified Aβ-sdAb 1a was further characterized by ELISAand immunohistochemical staining of tissues from transgenic mice andhuman patients.

4. ELISA Results for Single-Chain Aβ-sdAb 1a

FIG. 6 depicts the results of ELISA performed in Pierce's MaxiSorpplate, which had been coated with 500 ng/well of Aβ-42 peptide at pH 9.5overnight at room temperature. After washing the plate with water, theantigen coated wells were blocked with 1% BSA and subsequently treatedwith identical concentrations of Aβ-sdAb 1a and mouse-Aβ-mAb for thesame length of time and temperature (2 hours at RT). Detection was doneusing HRP labeled secondary antibody and TMB as a substrate. The bluecolor generated by HRP reaction was quenched with 1.0 M HCl and OD450recorded on Molecular Devices SpectraMax Plus plate reader. FIG. 6 isthe plot of OD450 readings of the chromogenic yellow color generated bythe reaction of the substrate with the HRP-enzyme. The single-domainantibody 1a clearly outperformed the commercial Aβ-mouse-mAb.

E. Administration and Dosage

Pharmaceutical formulations of a therapeutically effective amount of apolypeptide of the invention can be administered orally or parenterallyin admixture with a pharmaceutically acceptable carrier adapted for theroute of administration.

Methods well known in the art for making formulations are found, forexample, in Remington's Pharmaceutical Sciences (18th edition), ed. A.Gennaro, 1990, Mack Publishing Company, Easton, Pa. Compositionsintended for oral use may be prepared in solid or liquid forms accordingto any method known to the art for the manufacture of pharmaceuticalcompositions. The compositions may optionally contain sweetening,flavoring, coloring, perfuming, and/or preserving agents in order toprovide a more palatable preparation. Solid dosage forms for oraladministration include capsules, tablets, pills, powders, and granules.In such solid forms, the active compound is admixed with at least oneinert pharmaceutically acceptable carrier or excipient. These mayinclude, for example, inert diluents, such as calcium carbonate, sodiumcarbonate, lactose, sucrose, starch, calcium phosphate, sodiumphosphate, or kaolin. Binding agents, buffering agents, and/orlubricating agents (e.g., magnesium stearate) may also be used. Tabletsand pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and soft gelatincapsules. These forms contain inert diluents commonly used in the art,such as water or an oil medium. Besides such inert diluents,compositions can also include adjuvants, such as wetting agents,emulsifying agents, and suspending agents.

Formulations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, or emulsions. Examples of suitablevehicles include propylene glycol, polyethylene glycol, vegetable oils,gelatin, hydrogenated naphthalenes, and injectable organic esters, suchas ethyl oleate. Such formulations may also contain adjuvants, such aspreserving, wetting, emulsifying, and dispersing agents. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for the agents of the invention include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes.

Liquid formulations can be sterilized by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions, or by irradiating or heating the compositions.Alternatively, they can also be manufactured in the form of sterile,solid compositions, which can be dissolved in sterile water or someother sterile injectable medium immediately before use.

The amount of active polypeptide in the compositions of the inventioncan be varied. One skilled in the art will appreciate that the exactindividual dosages may be adjusted somewhat depending upon a variety offactors, including the ingredient being administered, the time ofadministration, the route of administration, the nature of theformulation, the rate of excretion, the nature of the subject'sconditions, and the age, weight, health, and gender of the patient. Inaddition, the severity of the condition targeted by an agent of theinvention will also have an impact on the dosage level. Generally,dosage levels of an agent of the invention of between 0.1 μg/kg to 100mg/kg of body weight are administered daily as a single dose or dividedinto multiple doses. Preferably, the general dosage range is between 250μg/kg to 5.0 mg/kg of body weight per day. Wide variations in the neededdosage are to be expected in view of the differing efficiencies of thevarious routes of administration. For instance, oral administrationgenerally would be expected to require higher dosage levels thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, which are well known in the art. In general, the precisetherapeutically effective dosage will be determined by the attendingphysician in consideration of the above-identified factors.

Methods for administering peptides to a subject are described, forexample, in U.S. Pat. Nos. 5,830,851; 5,558,085; 5,916,582; 5,960,792;and 6,720,407, hereby incorporated by reference.

F. Ex-Vivo and In-Vivo Results of Peptide Compositions 1A and 2A inAlzheimer's Disease Models

1. Detection of Amyloid Plaque in Transgenic Mouse and Human Alzheimer'sPatients with Peptide Composition 2A in Ex-Vivo Experiments

The specificity of Aβ-sdAb for A≈ was tested by immunohistochemical(IHC) staining of paraffin embedded brain tissues from the APPtransgenic mouse (FIG. 7A upper two frames) and Alzheimer's patient(FIG. 7A lower two frames), with and without primary Aβ-sdAb. The sametissues were stained with Thioflavin-S dye (FIG. 7B). Paraffin tissueswere cut in a microtome to the thickness of 5 microns, mounted on APEScoated slides, dried at room temperature (RT) for 24 hours, and thendeparaffinized using xylene and ethanol. Washed slides were blocked in10% normal serum with 1% BSA (2 hours at RT), and treated withsingle-chain Aβ-sdAb 2a in PBS containing 1% BSA (1:100 dilution,overnight at 4° C.). After washing slides with 0.1% Triton X-100,endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide,followed by treatment with biotinylated goat-anti-llama-IgG Detectionwas done with streptavidin-HRP and diaminobenzidine as HRP substrate.

2. Demonstration of BBB Permeability of Aβ-Vab Peptide Compositions 1aand 2a in Alzheimer's-Like Transgenic Mice

We tested the BBB permeability of Aβ-sdAb 1a and single-chain Aβ-sdAb2a. The single-chain Aβ-sdAb 2a had been prepared by the TCEP(triethoxy-phosphine) reduction of Aβ-sdAb 1a above in Section B.1. Todemonstrate BBB penetration, 60 ug of Aβ-sdAb 1 or single-chain Aβ-sdAb2a was injected in the tail vein (FIG. 8) of the live transgenic mice(J9 strain: PDGF-APP-SW-Ind; APP: amyloid precursor protein; SW and Indstand for Swedish and Indiana mutations in APP), according to theprotocol outlined below (Table 8). The commercial mouse-Aβ-mAb was alsoused for comparative purposes. Non-transgenic mice were used as negativecontrols.

TABLE 8 Protocol for Demonstrating BBB Permeability Number Group MouseAntibody Time of mice 1 APP tg mice ICBI-antibody 4 h 6 2 APP tg miceICBI-antibody 24 h 6 3 APP tg mice Mouse-Ab-IgG 4 h 6 4 APP tg miceMouse-Ab-IgG 24 h 6 5 Non-tg control mice ICBI-antibody 4 h 3 6 Non-tgcontrol mice ICBI-antibody 24 h 3 7 Non-tg control mice Mouse-Ab-IgG 4 h3 8 Non-tg control mice Mouse-Ab-IgG 24 h 3 APP = Amyloid precursonprotein. Route = Tail vein, Dose = 60 ug, Treatment and duration =variable.

Mice were sacrificed 4 hours and 24 hours after the injection and theirbrains serially sectioned. The two hemispheres were separated; the lefthemisphere was rapidly snap frozen on dry ice (2 to 5 min) and stored at−80° C.; the right hemisphere was immersed in a cold 4%paraformaldhehyde fixative solution.

3. Neuropathological Analysis

The fixed half brain was serially sectioned sagitally with the vibratomeat 40 um and stored at −20° C. in cryoprotective medium. Sections wereimmunostained with biotinylated anti-llama-IgG1 and detected withstreptavidin-HRP using an enzyme substrate, followed by imaging with thelaser confocal microscope (FIG. 9). Co-localization studies betweenllama IgG and Aβ-protein were also performed by staining the tissueswith Thioflavin-S dye. Digital images were analyzed with the ImageQuantprogram to assess numbers of lesions.

4. Results of Blood-Brain Permeability of Peptide Compositions

All six transgenic mice analyzed 24 hours after a single low doseinjection of 60 ug amyloid sd-antibody displayed labeling ofamyloid-plaque in their central nervous system. The data shown in FIG. 9represents data obtained only with single-chain Aβ-sdAb 2a. Binding ofpeptide compositions 1a and 2a to amyloid plaque was only detected inthe APP transgenic mice, not in non-transgenic mice. More importantly,Aβ-sd-antibodies 1a and 2a labeled both the soluble/diffusible plaqueand insoluble plaque, while Thioflavin-S dye labeled primarily theinsoluble plaque. Soluble plaque is the one responsible for cognitivedecline from Alzheimer's Disease, not the insoluble plaque, which islabeled by other neuroimaging agents such as Pittsburgh Compound B and¹⁸F-Flutemetmol. The single-chain sdAb 2a stained about 10% of all thesoluble and insoluble plaque in the mouse brain, while Aβ-sdAb 1alabeled about 3.6% of the total plaque in the same amount of time.

5. Pharmacokinetics Study of Single-Chain Aβ-sdAb 2a for Alzheimer'sDisease in Mice

A pharmacokinetics (PK) study of the single-chain Aβ-sdAb 2a wasconducted in collaboration with Biotox Sciences, San Diego. In thisstudy, three groups of mice (average weight: ˜25 g) were injected in thetail vein with 60 ug of single-chain Aβ-sdAb 2a in 200 ul of PBS buffer.At a predetermined timepoint, blood was drawn from the animals the serumwas analyzed for the concentration of the single-chain Aβ-sdAb 2a byELISA. All three sets of animals showed identical clearance of thesingle-chain Aβ-sdAb 2a from the blood (FIG. 10). FIG. 10 is a graph ofthe serum retention time of the single-chain Aβ-sdAb 2a in Aβ-mice. TheX-axis represents time in hours and the Y-axis concentration ofsingle-chain Aβ-sdAb 2a per ml of serum. The two broken lines indicatenon-linearity in the X-axis. Clearance of amyloid-plaque by binding tomouse-Aβ-mAb and subsequent phagocytosis has been reported in theliterature [Wang, Y-J, et al., Clearance of amyloid-beta in Alzheimer'sdisease: progress, problems and perspectives, Drug Discovery Today, 11,931 (2006)].

Although at 24 h the serum concentration of 2a in FIG. 10 dropped toabout half compared to what it was at 0.5 hour, its levels stayed at˜40% for 7 days, suggesting that the single-chain Aβ-sdAb 2a has a serumlife of at least 7 days and, therefore, a remarkable potential fordeveloping diagnostic and long-acting therapeutic agents. The slowdecrease in serum concentrations of the single-chain Aβ-sdAb 2a in thefirst 24 h could be attributed to its binding with the amyloid-peptide.

G. Synthesis of Antibody-Coated Nano-Particles with Peptide Compositions1 and 2 from FIG. 2

1. Synthesis of Polybuytcyanoacrylate (PBCA) Naoparticles 5

To overcome the shortcomings of the prior art, this invention describesthe synthesis of biodegradable polyalkylcyanoacrylate nanoparticlescoated with aminated dextran and peptide compositions 1 and 2 in FIG. 2.The synthetic steps are outlined in FIGS. 11-14. To a stirring solutionof aminated dextran 4 (1.0 gm) in 100 ml of 10 mM HCl (pH 2.5) wasslowly added 1 ml of butylcyanoacrylate (BCA) (FIG. 11 and FIG. 13). Thereaction mixture was allowed to stir at RT for 4 hours to obtain a whitecolloidal suspension, which was carefully neutralized with 0.1 M NaHCO₃solution to pH 7.0. This colloidal suspension was filtered through 100um glass-fiber filter to remove large particles. The filtrate was splitinto 50 ml centrifuge tubes and spun at 10,000 RPM for 45 minutes. Afterdiscarding the supernantant, the particles were washed several timeswith deionized water, centrifuging the particles and discarding thesupernatant until no more white residue was seen in the supernatant. Theresulting PBCA particles, 5, were stored in 0.01% NaN₃/PBS at 4° C.(FIG. 11 and FIG. 13).

2. Synthesis of Thiolated PBCA Naoparticles 6

PBCA particles 5 were washed with 50 mM MOPS buffer, pH 7.0, to removeNaN₃. The particles were then treated 50 mM Traut's regeant in MOPSbuffer for one hour to synthesize thiolated PBCA nanoparticles 6 (FIG.11 and FIG. 13). The particles were then repeatedly washed to remove theunreacted Traut's reagent.

3. Synthesis of Peptide Composition Maleimido Derivatives 3 and 8

Purified peptide composition 1a (1 mg, 12.5 nM) was dissolved in 50 mMMOPS buffer, pH 7.0. It was treated with NHS-PEG-Mal (MW: 3000 Da)(250nM) at RT for 1 hour (FIG. 12). The reaction was concentrated onAmicon-Centricon Concentrators (MW Cutoff: 30 KDa) to remove hydrolyzedand unconjugated excess NHS-PEG-Mal. The purified pegylated derivative 8was characterized by MALDI-MS and 12% SDS-PAGE gel. A similar processcan be used to convert peptide composition 2 in FIG. 2 into thepegylated derivative 3 (FIG. 11).

4. Synthesis of Covalently Conjugated Peptide Composition Nanoparticles7 and 2

The conjugation of maleimido-Aβ-sdAb 8 with thiolated PBCA nanoparticles6 was carried out at pH 7.0 in 50 mM MOPS buffer in the presence of 5 mMEDTA for 4 hours at RT (FIG. 14). The resulting PBCA nanoparticles 9were purified by washing off (5×50 ml deionized water) the unreactedmaleimido antibody 3. The particles were stored in deionized water at 4°C. until used. A similar process can be used to convert pegylatedderivative 3 and thiolated PBCA nanoparticles 6 into PBCA nanoparticles7 (FIG. 12).

The present disclosure is not to be limited in terms of the particularembodiments described in this application. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods, processes and compositions within the scope of the disclosure,in addition to those enumerated herein, will be apparent to thoseskilled in the art from the foregoing descriptions. Such modificationsand variations are intended to fall within the scope of the appendedclaims. The present disclosure is to be limited only by the terms of theappended claims, along with the full scope of equivalents to which suchclaims are entitled. It is to be understood that this disclosure is notlimited to particular methods, processes, reagents, compoundscompositions or biological systems, which can of course vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

All publications, patent applications, issued patents, and otherdocuments referred to in this specification are herein incorporated byreference as if each individual publication, patent application, issuedpatent, or other document was specifically and individually indicated tobe incorporated by reference in its entirety. Definitions that arecontained in text incorporated by reference are excluded to the extentthat they contradict definitions in this disclosure.

While certain embodiments have been illustrated and described, it shouldbe understood that changes and modifications could be made therein inaccordance with ordinary skill in the art without departing from thetechnology in its broader aspects as defined in the following claims.

1.-96. (canceled)
 97. A composition comprising: a polypeptide comprisingthe formula R1-L2-R4-Y-R3-X-R5-L1-R2; wherein R1 comprises a variableantigen-binding domain from a camelid single-domain heavy chain antibodycomprising amino acids 1-127 of the amino acid sequence of SEQ ID NO: 11and R2 is selected from a constant domain CH1, CH2 or CH3 of human IgG,a constant domain CH2 or CH3 of camelid IgG, or constant domain CH1,CH2, CH3, CH4 or CH5 of shark IgNAR; wherein L1 and L2 each comprise ahinge region from a camelid single-domain heavy-chain only antibody;wherein X and Y are bifunctional linkers, selected from amaleimido-thiol conjugate and polyethylene glycol; wherein R3 comprisesat least one constant domain of human IgG CH2 or CH3 domain; wherein R4and R5 are selected from proteins that bind to receptors on endothelialcells that form the blood-brain barrier.
 98. The composition of claim108, wherein the R4 or R5 is a FC5, FC44, insulin, transferrin, Apo-B,Apo-E, Apo-E4, wherein the polypeptide can cross the blood-brainbarrier.
 99. The composition of claim 108, wherein the R4 or R5 is IL17,IL22.
 100. The composition of claim 108, wherein the R4 or R5 is atherapeutic agent, protein, peptide, or an enzyme.
 101. The compositionof claim 108, wherein the nanoparticle has a molecular weight of about70 kDa.
 102. The composition of claim 108, wherein a hinge regioncorresponds to the amino acid sequence of SEQ ID NO:
 13. 103. Thecomposition of claim 108, wherein at least one of L1 and L2 iscovalently conjugated to—NH—C(═NH2.HCl)-CH2CH2CH2-S-Maleimide-PEG-CONH—, which in turn isconjugated to a nanoparticle.
 104. A method for introducing a targetpeptide in the central nervous system of a subject, the methodcomprising administering to the subject a polypeptide of claim 108.